WO2007001080A1 - Beverage/food and pharmaceutical comprising loquat leaf extract - Google Patents

Abstract

A beverage/food or pharmaceutical comprising a loquat leaf or an extract or purified product from a loquat leaf as an active ingredient and has one or more effects selected from the group consisting of an antihyperlipemic effect, an antihypertensive effect, an inhibitory effect on the proliferation of a cancer cell, an effect of inducing the apoptosis of a cancer cell, an effect of producing an active oxygen species, an antihyperglycemic effect and an anti-oxidant effect.

Description

 Food / beverage products and pharmaceuticals containing bi-leaf extract

 The present invention includes, for example, an antihyperlipidemic action, an antihypertensive action, a cancer cell proliferation inhibiting action, a cancer cell apoptosis inducing action, a reactive oxygen species producing action, a hyperglycemic lowering action and / or an antioxidative action or The present invention relates to pharmaceuticals and their manufacturing methods. Technical background

 Bean leaves are used as a favorite drink of bean tea and are not only popular as bean tea but also used as a medicine blended in traditional Chinese medicine.

 In recent years, the functionality of birch leaves has been revealed. For example, the cytotoxic activity of polyphenol components contained in birch leaves against human tumor cells (Non-patent Document 1), and the anti-carcinogenic promotion effect of Megastigman glycosides contained in birch leaves (Non-Patent Documents) 2), the antitumor activity (non-patent document 3) and anti-oxidant action (non-patent document 4) of birch leaves have been reported.

 Patent Document 1 filed by the present applicant discloses a method and production equipment for producing a large amount of flavored bean tea using bean leaves. The present applicant uses the bean-cha tea manufacturing facility described in Patent Document 1 to produce screw mebiwa tea by roasting tourmaline stone. Concerning the manufactured Nemebiwa tea, consumers say that “the blood sugar level of diabetes has decreased” and “the blood pressure of hypertension has returned to normal”, but empirical evidence has been confirmed. (Non-Patent Document 5).

 On the other hand, the paragraph number “0 0 0 4” of Patent Document 1 states that “The leaves of bean koji, which is a material for producing conventional koji tea, use the cultivated varieties with the aim of harvesting the fruits. In the past, there was a disadvantage that the original effect was not obtained even if it was used for medicinal purposes because the nutrients collected in the fruit and not enough nutrients were distributed to the birch leaves. Was not sufficiently effective for medicinal purposes.

Conventionally, it has been disclosed that bean has the following activities or usage methods. ing.

 Patent Document 2 discloses that koji or an extract thereof has a matrix meta-mouth proteinase (MMP-1) inhibitory activity.

 Patent Document 3 discloses that the leaves of birch trees are baked into charcoal to make a powdered food material.

 In Patent Document 4, the raw leaves of bean paste are cut, steamed with steam, squeezed while sending hot air, dried, shaped, and then dried again to increase the aroma and taste. A characteristic method for producing Kampo tea is disclosed.

 Patent Document 5 discloses a method for producing health tea, which is obtained by mixing leaf green health tea of Gramineae plant and * leaf of birch and containing more γ-aminobutyric acid.

 Patent Document 6 discloses that a koji extract excluding a fruit part has a sarcoidase inhibitory activity.

 Patent Document 7 discloses a composition for promoting nourishing tonic health in which shakiyota and bibi leaf are mixed.

 Patent Document 8 discloses a health tea obtained by mixing roasted yellow persimmon leaves with bean tea.

 Patent Document 9 discloses a health tea composed of a mixture of sweet tea and bean paste. Patent Document 1 Q discloses that a composition containing an organic extract of the genus Rosaceae inhibits C0X-2 activity in living organisms. Patent Document 1 Japanese Patent No. 3452351

Patent Document 2 JP 2005-008539 A

Patent Document 3 Japanese Patent Application Laid-Open No. 2004-154108

Patent Document 4 JP 2004-105036 A

Patent Document 5 Japanese Patent Application Laid-Open No. 2002-0652 7

Patent Document 6 Japanese Patent Application Laid-Open No. 2001-163795

Patent Document 7 JP 2001-163792 A

Patent Document 8 Japanese Patent Application Laid-Open No. 7-274832

Patent Document 9 Japanese Patent Laid-Open No. 5-056772 Patent Document 1 o Special Table 2004-529079

Non-Patent Document 1 Ito, H. et al., “Chem. Pharm. Bull.” 2000, pp. 48, p. 687-693 Non-Patent Document 2 Takashi Yoshida et al., “Bio Industry”, 2003, 20th 27, 33 Non-Patent Document 3 Ito, H. et al., “J. Agric. Food Chem.”, 2002, 50th, p. 2400-2403

Non-Patent Document 4 Jung, H. et al., “Arch. Pharm. Res. J, 1999, Vol. 22, p. 213-218 Non-Patent Document 5 Totsukawa Farm, Agricultural Production Corporation,“ Nemebiwa Tea Pamphlet ” , 2004 Invention Disclosure

 As described above, although various biological activities and active compounds contained in birch leaves have been clarified in the past, birch leaves have a hypoglycemic-lowering action, an antihypertensive action, and an inhibition of cancer cell growth. It has not been known to have effects such as action, cancer cell apoptosis-inducing action, reactive oxygen species producing action, and antihyperlipidemic action. If these effects can be confirmed, biliary leaves can be used for the prevention or treatment of diseases such as diabetes, hyperlipidemia, hypertension, and cancer.

 Therefore, in view of the above situation, the present invention uses an anti-hyperlipidemic action, a hypertension-inhibiting action, a cancer cell proliferation-inhibiting action, a cancer cell apoptosis-inducing action, a reactive oxygen species producing action, a hyperglycemia using biliary leaves. The object is to provide a food or drink or medicine having a lowering action and / or antioxidant action and a method for producing the same.

 As a result of intensive studies to solve the above-mentioned problems, the present inventors have reduced the blood glucose level by subjecting bean tea to hot water extraction and feeding the obtained extract to model mice or rats. It was also found that the weight of serum lipids such as cholesterol and triglycerides and adipose tissue is reduced. It was also found that the crude fraction obtained from the extract of bime tea has a strong antioxidant effect.

Furthermore, by applying bi ヮ cha to hot water extraction and feeding the resulting extract to model mice or rats, the increase in blood pressure is suppressed, and the growth is also caused by acting directly on cancer cells. It has been found that it suppresses and induces apoptosis of cancer cells and produces Z or reactive oxygen species, and has completed the present invention. The present invention includes the following.

 (1) Contains an extract or purified product of bime leaves or bime tea as an active ingredient, and also has an antihyperlipidemic effect, an antihypertensive effect, an anticancer effect on cancer cell proliferation, an inducement effect on cancer cell apoptosis, an active oxygen species A food or drink or a pharmaceutical product having one or more actions selected from the group consisting of a production action, a hypoglycemic action and an antioxidant action.

 (2) The food or drink or pharmaceutical product according to (1), wherein the bean tea is Nemebiwa tea.

 (3) The purified product is one or more selected from the group consisting of chlorogenic acid, quercetin 3-sambubioside, methyl chlorogenic acid, kaempferol 3-rhamnoside, quenorece'tin 3-rhamnoside, 2oc_hydroxyursolic acid and ursolic acid The food / beverage product or medicine according to (1), characterized in that it does not contain a compound of

 (4) The above food or drink is selected from the group consisting of antihyperlipidemic action, antihypertensive action, cancer cell proliferation inhibitory action, cancer cell apoptosis inducing action, reactive oxygen species production action, hyperglycemia lowering action and antioxidant action. The food / beverage product or pharmaceutical product according to (1), wherein the food / beverage product or the pharmaceutical product has a label indicating that it has at least one action.

 (5) An anti-sebaceous action, an antihypertensive action, an antihypertensive action, a cancer cell growth inhibitory action characterized by comprising a step of subjecting the birch leaf or bibi tea to hot water extraction or solvent extraction to obtain an extract, A method for producing a food or drink or pharmaceutical having one or more actions selected from the group consisting of cancer cell apoptosis-inducing action, reactive oxygen species producing action, hyperglycemic lowering action and antioxidant action.

 (6) The production method according to (5), further comprising a step of subjecting the extract to a purification means to obtain a purified product.

 '(7) The production method according to (6), wherein the purification means is column chromatography. .

(8) The manufacturing method according to (5), wherein the bean tea is Nememebiwa tea. '

(9) The purified product is selected from the group consisting of chlorogenic acid, taercetin 3-sambubioside, methyl chlorogenic acid, kaempferonole 3-rhamnoside, quercetin 3-rhamnoside, 2α-hydroxyursolic acid and ursolic acid 1 Above (6) The production method according to (6), wherein the compound is not contained.

 Hereinafter, the present invention will be described in detail.

The food / beverage product or pharmaceutical product according to the present invention contains an extract or purified product of bean leaf or bean tea as an active ingredient, and has an antihyperlipidemic effect, an antihypertensive effect, an anticancer effect on cancer cell growth, It has one or more actions selected from the group consisting of cancer cell apoptosis-inducing action, reactive oxygen species producing action, hyperglycemic lowering action and antioxidant action. Hyperlipidemia and hypertension can be prevented or treated by ingesting or administering the food or drink or pharmaceutical product according to the present invention to animals such as humans. In addition, by ingesting or administering the food or drink or medicine according to the present invention to animals such as humans, the growth of cancer cells is suppressed, apoptosis is induced in cancer cells, and reactive oxygen species are produced in cancer cells. It can be shown. Furthermore, by ingesting or administering the food or drink or medicine according to the present invention to animals such as humans, the blood glucose level can be lowered and active oxygen or the like can be removed. Here, the antihyperlipidemic effect means that the cholesterol in the blood is decreased. Also, the antihypertensive activity, means to suppress the increase of the 'blood pressure _ς

 The cancer cell growth inhibitory action means killing cancer cells and suppressing their growth. The cancer cell apoptosis-inducing action means that cancer cells are induced to induce apoptosis and kill the cancer cells. Further, the reactive oxygen species producing action means producing reactive oxygen species in cancer cells and killing the cancer cells.

 Further, the hypoglycemic action means that the blood sugar level is lowered. Furthermore, antioxidation means removing active oxygen in the body.

In the present invention, koji leaf or koji tea is used. As bibi leaves, fresh leaves or dried leaves can be used as they are. As the bean tea, any bean tea can be used, but for example, Nejimebiwa tea (trade name), _σ Nemebiwacha is a product that is more scorched by high-temperature heating with tourmaline stone. It can be prepared by the method described in Patent Document 1 described above. In addition, Nemebiwa tea (Neiso-cha) is sold from Totsukawa Farm, Kagoshima Agricultural Production Corporation.

The extract used for the food or drink or the pharmaceutical product according to the present invention can be obtained by subjecting the birch leaves or bibi tea to hot water extraction or solvent extraction. For example, bean leaves or bean tea Is subjected to extraction using hot water, and this is repeated once or several times (for example, 3 times) to obtain a bime leaf or bime tea extract. Alternatively, for example, bi-biba or bi-bicha extract can be obtained by subjecting bi-bipo or bi-bicha to extraction using water or an organic solvent such as lower alcohol Nyaseton. In the present invention, bi koji leaf or koji tea extract means an extract or various solvent extracts obtained by the above extraction method, a diluted solution thereof, a concentrated solution thereof or a dried powder thereof.

 Further, in the present invention, a purified product obtained by removing impurities from the extract by subjecting the above-described bibi leaf or bime tea extract to purification means such as filtration, centrifugation, or purification treatment is used. Can do. Examples of purification means include column chromatography, normal phase or reverse phase chromatography, ion exchange chromatography, and gel filtration, with column chromatography being particularly preferred.

 For example, an aqueous solution of bime leaf or bime tea is directly subjected to open column chromatography on a gel for reverse phase (eg, MCI gel CHP-20P (Mitsubishi Chemical), etc.). Chromatography is performed by using water as the liquid A and ethanol as the liquid B in the mobile phase, changing the ratio of the liquid A and the liquid B to 100: 0, 30:70, 70:30. Finally, use acetonitrile as solution C, and perform chromatography with the ratio of solution A and solution C set to 50:50. The crude fraction that elutes at a ratio of A to B of 70:30 and the crude fraction that elutes at a ratio of 50 to 50 of A and C shows a strong hypoglycemic action. . Therefore, these crude fractions can be suitably used for foods and beverages or pharmaceuticals according to the present invention as purified products of piu leaves or boiled tea.

 Alternatively, after the solvent is removed from the extract solution of bean leaf or bean tea extracted with 80% permanent caseon using a vacuum evaporator, the residue is converted into a gel for reverse phase (for example, MCI gel CHP-2

OP (Mitsubishi Chemical) and other open column chromatography. Chromatography using liquid gradient from liquid A to liquid B using water as liquid A and methanol as liquid B as the mobile phase. Finally, use Acetone as solution C, and perform chromatography with the ratio of solution A and solution C set to 50:50. The crude fraction eluted with this method has a strong antioxidant effect. Therefore, this crude fraction can be suitably used for a food or drink or a pharmaceutical product according to the present invention as a purified product of bean leaf or bean tea. In this crude fraction, chlorogenic acid, quercetin 3-sanbubio It does not contain one or more of the following compounds: sid, methylcuccinic acid, kenwei chinolose 3-rhamnoside, quercetin 3-rhamnoside, 2-hydroxyursolic acid and ursolic acid. Therefore, it is preferable that these compounds are not contained in the koji leaf or koji tea purified product used for the food or drink or the pharmaceutical product according to the present invention.

 By using the koji leaf or koji tea extract or purified product described above as an active ingredient, the food or drink or the pharmaceutical product according to the present invention can be produced.

 By adding an effective amount of the extract or purified product of the present invention to any form such as tablets, capsules, granules, drinks, pet bottles, etc., or adding it to any food, the food and drink according to the present invention Goods can be obtained. The food / beverage product according to the present invention has an antihyperlipidemic action, an antihypertensive action, a cancer cell proliferation inhibitory action, a cancer cell apoptosis inducing action, a reactive oxygen species producing action, a hyperglycemic lowering action and / or an antioxidant action. It can be used as a food and drink, especially as a health supplement or food for specified health use. Preferably, it is a health supplement or special health food in the form of tablets, 'capsules, granules, drinks, PET bottles, etc.

 Examples of food and drink include, but are not limited to, confectionery, retort food, juices, teas, and dairy products. In addition, sweeteners, seasonings, emulsifiers, suspending agents, preservatives, etc. may be added to foods and drinks as necessary, or vitamins, nutrients, immune enhancers (for example, propolis) You can add mushroom extract etc.).

 The amount of the extract or purified product of bime leaves or bime tea to the food and drink according to the present invention is an amount within a range corresponding to 0.1 to 200 mg per kg of adult body weight to be ingested or 50 mg to 1 item. lg, but not limited to this range.

 On the other hand, the pharmaceutical product according to the present invention contains an effective amount of an extract or a purified product of bime leaves or bime tea. The pharmaceutical agent according to the present invention is used as an antihyperlipidemic agent, a hypertension inhibitor, a cancer cell proliferation inhibitor, a cancer cell apoptosis inducer, a reactive oxygen species producing agent, a hyperglycemic agent or an antioxidant. Can do.

The medicinal product according to the present invention includes, in addition to an extract or purified product of bean leaf or bean tea, a pharmaceutically acceptable carrier (excipient or diluent), a binder, a bulking agent, and a lubricant. Disintegrating agents, wetting agents, emulsifying agents, buffering agents, suspending agents, preservatives, coloring agents, flavoring agents and An additive appropriately selected from sweeteners and the like can be contained. Carriers and additives that are generally used for formulation can be used for the production of the pharmaceutical product according to the present invention. For example, examples of the binder include starch, polyvinyl chloride, hydroxypropylmethylcellulose, and the like. Examples of bulking agents include ratatoose and microcrystalline cellulose. Examples of lubricants include talc, silica, and magnesium stearate. Examples of disintegrants include starch and sodium starch glycolate. Examples of wetting agents include sodium lauryl sulfate. Examples of emulsifiers include cell mouth derivatives, sorbitol and the like. Examples of preservatives include methyl-P-hydroxybenzoate and sorbic acid. However, the additives that can be used in the present invention are not limited to these additive examples.

 The pharmaceutical product according to the present invention is formulated for oral administration or parenteral administration (intravenous, intraarterial, intraperitoneal, intrarectal, subcutaneous, intramuscular, sublingual, intranasal, intravaginal, etc.) Can be. The form of the preparation is not particularly limited, and examples thereof include solutions, tablets, powders, granules, capsules, suppositories, sprays, controlled release agents, suspensions and drinks. '

 The dose of the extract or purified product of bean leaf or bean tea contained in the pharmaceutical product according to the present invention may vary depending on factors such as the patient's age, weight, sex, condition and severity. The daily dose of the extract or purified product of bicucumber leaf or bibi tea administered to a patient is, for example, in the range of 0.1 to 200 mg, preferably 1 to 100 mg per kg of patient weight. It is not limited to. If necessary, the dose may be divided into several doses, for example, divided into 2-3 doses. In addition, the pharmaceutical agent according to the present invention may have other antihyperlipidemic agents having the same or different therapeutic uses, hypertension inhibitors, cancer cell proliferation inhibitors, cancer cell apoptosis inducers, reactive oxygen species producing agents, hyperglycemic agents. And / or can be administered to a patient in combination with an antioxidant. The food / beverage products or pharmaceutical products according to the present invention can be subjected to pharmacological evaluation as follows, for example.

Examples of the pharmacological evaluation of the antihyperlipidemic action of the food and drink or pharmaceutical product according to the present invention include, for example, ingesting the food and drink or pharmaceutical product according to the present invention in a model animal and A method for measuring tissue weight is mentioned. The present invention When the amount of total cholesterol, HDL cholesterol and / or triglyceride in the blood is significantly reduced in the animals receiving the food / drinks / medicines according to the present invention compared to the animals not taking the food / drinks / medicines Therefore, it can be determined that the food or drink or pharmaceutical product according to the present invention has an antihyperlipidemic effect. Similarly, when the adipose tissue weight of the organ is significantly reduced, it can be determined that the food or drink or the pharmaceutical product according to the present invention has an antihyperlipidemic effect well.

 In addition, examples of the pharmacological evaluation of the antihypertensive action of foods and beverages or pharmaceuticals according to the present invention include a method using males of SHR rats (natural hypertension rats). In other words, the food or beverage or pharmaceutical product according to the present invention is fed to the SHR rat, and after the breeding is finished, the blood pressure of each rat is measured using an unheated cuff blood pressure meter (Muromachi Kikai Co., Ltd.). When the blood pressure is reduced as compared with a rat not fed with the food or drink or pharmaceutical product according to the present invention as measured by the apparatus, the food or food product or pharmaceutical product according to the present invention has a good antihypertensive product. I can judge.

 Furthermore, the pharmacological evaluation of the cancer cell 'growth-inhibiting action, cancer cell apoptosis-inducing action and reactive oxygen species producing action of the food or drink or pharmaceutical product according to the present invention includes, for example, human acute

'Method using promyelocytic leukemia disease cells (HL-60 cells). That is, when HL-60 cells are cultured in the presence of a food or drink or pharmaceutical product according to the present invention and compared with the control group (in the absence of the food or food product or pharmaceutical product according to the present invention), It can be judged that the food or drink or the pharmaceutical product according to the present invention has a cancer cell growth inhibitory effect. In addition, HL-60 cells were cultured in the presence of the food or drink or pharmaceutical product according to the present invention, and its 50% survival rate was suppressed compared to the control group (in the absence of the food or food product or pharmaceutical product according to the present invention). In this case, it can be determined that the food or drink or pharmaceutical product according to the present invention has a cancer cell apoptosis-inducing action. Furthermore, when HL-60 cells are cultured in the presence of a food or drink or pharmaceutical product according to the present invention, and the intracellular active oxygen is increased compared to the control group (in the absence of the food or food product or pharmaceutical product of the present invention) Therefore, it can be judged that the food or drink or the pharmaceutical product according to the present invention has a reactive oxygen species producing action on cancer cells.

In addition, as a pharmacological evaluation of the hypoglycemic action of the food or drink or pharmaceutical product according to the present invention, for example, a model animal (for example, a KKAy mouse that is a type II diabetes model mouse) is ingested. Blood glucose level during or after breeding The method to determine is mentioned. When the blood glucose level is significantly reduced in the animal that has taken the food or drink or medicine according to the present invention, compared to the animal that has not taken the food or drink or medicine according to the present invention, the food or drink according to the present invention Alternatively, it can be determined that the drug has good hypoglycemic action. Similarly, in a glucose tolerance test using a model animal, if the blood glucose level or HbAlC value significantly decreased in an animal that had consumed the food or drink or pharmaceutical product according to the present invention, the food or drink product or pharmaceutical product according to the present invention was It can be judged that it has a good hypoglycemic effect.

 Furthermore, examples of the pharmacological evaluation of the antioxidant action of foods and beverages or pharmaceuticals according to the present invention include the method described in Harwat, KSM et al., Free Radical Res., 36: 177-187, 2002. . That is, DPPH (1, ldiphenyl-2-picrylhydrazyl) is added to the food / beverage product or pharmaceutical product according to the present invention, the absorbance is measured, and the concentration of TrQlox is determined from the absorbance using the Trolox absorbance standard curve. Is converted. From the converted value, the radical scavenging rate is calculated from the 'Trolox radical scavenging standard curve. When the radical scavenging rate of the food or drink according to the present invention is equal to or higher than, for example, the positive control vitamin C or E, the food or drink according to the present invention has a good antioxidant effect. It can be judged that it has.

 Non-roasted bean leaves are anti-hyperlipidemic, antihypertensive, cancer cell growth inhibitory, cancer cell apoptosis-inducing, reactive oxygen species producing, and hypoglycemic action Or, no antioxidant action is observed. On the other hand, in the present invention, by using an extract or purified product of bean koji leaf or koji tea, an anti-lipidemia action, an antihypertensive action, a cancer cell proliferation inhibiting action, a cancer cell apoptosis inducing action, an active oxygen It is possible to produce a food or drink or a pharmaceutical product having a seed production action, a hyperglycemic action and / or an antioxidant action. In other words, the foods and medicines used are useful for the prevention or treatment of diseases such as diabetes, hyperlipidemia, hypertension and cancer. -This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2005-187133, which is the priority basis of the present application. Brief Description of Drawings

Fig. 1 shows rats in the control group and the bi ヮ tea group during the breeding period in Example 1. The result of having investigated the weight transition of is shown.

 FIG. 2 shows the results of examining the total cholesterol level in the blood of the rats of the control group and the bime tea group in Example 1.

 FIG. 3 shows the results of examining the amount of HDL cholesterol in the blood of the rats in the control group and the bime tea group in Example 1.

 FIG. 4 shows the results of examining the amount of triglyceride in the blood of the rats of the control group and the green tea group in Example 1.

 FIG. 5 shows the results of measuring the weights of the liver, the peripheral testicles, and the adipose tissue around the kidneys of the rats in the control group and the bicha tea group after the breeding in Example 1.

 FIG. 6 shows the results of examining the body weight changes of the mice in the control group and the bi ヮ tea group during the breeding period in Example 1. ·

 FIG. 7 shows the results of measurement of the weight of the fat tissue around the kidney and the accessory testicles of the mice in the control group and the birch tea group after the breeding in Example 1.

 FIG. 8 shows changes in blood glucose levels of mice in the control group and the bi ヮ tea group during the breeding period in Example 2.

 FIG. 9 shows the results of the glucose tolerance test of mice in the control mouth group and the bibi tea group after breeding in Example 2.

 FIG. 10 shows the measurement results of blood HbAlC values in mice of the control group and the bi ヮ tea group after the glucose tolerance test in Example 2.

 FIG. 11 shows a thin-layer chromatograph of the dried crude fraction in Example 3. FIG. 12 shows the transition of the blood glucose level of the mice in the control group and each rice bran and tea group during the breeding period in Example 3. FIG. 13 shows the results of the glucose tolerance test of the mice in the control group after breeding in Example 3 and in the bi ヮ tea group fed with fractions 3 and 4. ·-Fig. 14 shows the measurement results of blood HbAlC values in mice of the control group and each of the bicha tea groups after the glucose tolerance test in Example 3.

 FIG. 15 shows a thin-layer chromatograph of the crude fraction in Example 4.

FIG. 16 shows the results of examining the antioxidant action of the crude fraction in Example 4. Figure 17 shows the antioxidant activity of the crude fraction prepared in Example 3 in Example 5. The results of the investigation are shown.

 FIG. 18 shows the results of examining the antioxidant action of the crude fraction further obtained from fraction 3 of Example 3 in Example 5.

 FIG. 19 shows the results of examining the antihypertensive effect of Nemebiwa tea extract in Example 6.

 FIG. 20 is a photograph of an agarose gel electrophoresis in Example 8, showing the results of examining the apoptosis-inducing action of the crude fraction of Example 3.

 FIG. 21 is a photograph of an agarose gel electrophoresis in Example 8, showing the results of examining the apoptosis-inducing action of the crude fraction further obtained from fraction 3 in Example 3.

 FIG. 22 shows the results of examining the effect of active oxygen species production on the cancer cells of the crude fraction of Example 3 in Example 9.

 FIG. 23 shows ligation obtained by examining the effect of reactive oxygen species production on cancer cells of the crude fraction further obtained from fraction 3 of Example 3 in Example 9. Best Mode for Carrying Out the Invention ''

 〔Example〕

 EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples. However, the technical scope of the present invention is not limited to these examples.

 In addition, the experiment of the antioxidant action in the following Example was performed using the method as described in the following literature (Harwat, K. S. M. et al., Free Radical Res., 36: 177-187, 2002). .

 Nemebiwa tea used in the following examples is commercially available from Totsugawa Farm, Kagoshima Agricultural Production Corporation. -In addition, the results in the figure accompanying each example are shown as the average value or average soil standard deviation of each group.

 [Example 1] Diet effect of Nemebiwa tea extract

 (1) Preparation of extract of Nemebiwa tea

r— Nejimebiwa tea (50 g) sold with hot water (400 ml). This was repeated three times. The obtained hot water extract was subjected to freeze-drying (Tokyo Rikakikai Co., LTD, Freeze Dryer FR-1) to obtain a dry powder.

 The total dry weight of Nemebiwa tea used was 346.2 g, and the total dry powder amount of the hot water extract was 57.37 g. Therefore, the dry powder content of the hot water extract relative to Nemebiwa tea was 16.57%.

(2) Animal preparation

 Wistar rats (Japan SLC) and KKAy mice (Claire Japan) were used. In the feeding test, the materials weighed so that the proportions shown in Table 1 below were mixed, kneaded with distilled water, and then dried in a freeze dryer. It was used as a food (a rat or a mouse fed with the food was referred to as a “control mouth group” in this example). Also, add the dry powder of the hot water extract obtained in (1 ') above to the control food so that it becomes 1%, and add this to the Nemebiwa tea group food (the rat or mouse fed with the food). In this example, it was used as “biso tea group”. table 1

(3) Rat serum lipid analysis and organ weight measurement

Experiments with 12 Wistar rats (Japan SLC) The group was divided into a control group (6 animals) and a bi ヮ cha group (6 animals), and the control group rats were allowed to freely take control food. Rats in the bi ヮ cha group were given free access to the Nemebiwa tea group. Both groups were raised for 1 month each.

 Rats were weighed every 3 days during the breeding period. In addition, both groups used tap water for the water supply of the rats, and the water supply was free during the breeding period.

 After the breeding, each animal was anesthetized with Nembutal, blood was collected from the heart, and serum lipids were analyzed. In addition, the organs of each group of rats were excised and weighed.

 Figure 1 shows the results of rat body weight measurement. Figure 1 shows the results of examining the weight transition of rats in both groups during the breeding period. In Fig. 1, the black circles are the results for the rats in the control group, and the white circles are the results for the rats in the bi ヮ cha group.

 As shown in Fig. 1, the weight of the rats in both groups increased during the breeding period, but a difference in weight gradually appeared between the two groups from the 3rd week. It can be seen that the increase is suppressed compared to the weight of the rat.

The analysis results of serum lipids after breeding are shown in Figs. Figure 2 shows the results of examining the total cholesterol content in the blood _(m g / dl). Figure 3 shows the results of examining the amount of HDL cholesterol in the blood (rag / dl). Figure 4 shows the results of examining the amount of triglyceride (mg / dl) in the blood. In FIGS. 2 to 4, “control” is the result for the rats in the control group, and “bi ヮ cha” is the result for the rats in the bi ヮ tea group.

 As shown in Figs. 2 and 3, it can be seen that the total cholesterol and HDL cholesterol levels are slightly lower in the rat tea group compared to the control group rats. For triglycerides, as shown in Fig. 4, the rats in the bi ヮ cha group should be moved at a lower level (* in Fig. 4 indicates P is 0.01) than in the control group. I understand. '' Further, the results of examining the organ weight after breeding are shown in FIG. Figure 5 shows the results of measuring the weight of rat liver, rat testicles, and adipose tissue around the kidney after the breeding. In FIG. 5, “control” is the result of the rats in the control group, and “b ヮ cha” is the result of the rats in the boiled tea group.

As shown in Fig. 5, as a result of comparing the weights of the adipose tissue around the liver, the epididymis, and the kidneys, the weight of the rats in the bicha tea group compared with the weight of the rats in the control group. It can be seen that gag is lower. In particular, with regard to the weight of adipose tissue around the kidney, it can be seen that the weight of rats in the bi ヮ cha group is significantly lower (* indicates P, 0.05 in Fig. 5).

 (4) Measurement of mouse body weight and organ weight:

 Experiments were performed using 20 KKAy mice (CLEA Japan). These mice were divided into a control group (10 animals) and a bime tea group (10 animals), and the control group mice were allowed to freely take control food. In addition, mice in the bi ヮ cha group were allowed to freely consume Nemebiwa tea group food. Both groups were raised for 7 weeks each.

 During the breeding period, mice were weighed every 3 days. 'In addition, both groups used tap water for the water supply, and the water supply was free during the breeding period.

 After the breeding, each animal was anesthetized with Nembutal, and the organs of each group of mice were removed and their weights were measured.

 The result of measuring the body weight of the mouse is shown in FIG. Figure 6 shows the results of examining the weight transition of mice in both groups during the breeding period. In Fig. 6, the black circles are the results for the control group of mice, and the white circles are the results for the Biwacha group of mice. In FIG. 6, * indicates P and 0.05, and ** indicates P and 0.01. '

 As shown in Figure 6, the weights of the mice in both groups increased during the breeding period, but the weight of the mice in the bi ヮ cha group increased significantly compared to the weight of the mice in the control group (P 01) It can be seen that it is suppressed.

 Fig. 7 shows the results of examining organ weights after breeding. Fig. 7 shows the results of measuring the weight of adipose tissue around the kidney and the accessory testicle after the breeding. In FIG. 7, “control” is the result of the mice in the control group, and “bi ヮ cha” is the result of the mice in the bi ヮ tea group.

 As shown in Fig. 結果, the weight of adipose tissue around the kidney and the vice testicles was compared.-The weight of the mouse in the bicha tea group was lower than that of the mouse in the control group. I understand. In particular, with regard to the weight of the adipose tissue around the kidney, it can be seen that the weight of mice in the bi ヮ cha group is significantly lower (* indicates P is 0.05 in Fig. 7).

[Example 2] High blood glucose level inhibitory effect of Nemebiwa tea extract (1) Preparation of extract of Nemebiwa tea

 Commercial Nemebiwa tea (50 g) was extracted with hot water (400 ml). This was repeated three times. The obtained hot water extract was freeze-dried (Tokyo Rikakikai Co., LTD, Freeze Dryer FR-1) to obtain a dry powder.

 The total dry weight of Nemebiwa tea used was 30.0 g, and the total dry powder amount of the hot water extract was 51.73 g. Therefore, the dry powder content of the hot water extract relative to Nemebiwa tea was 17.24%.

 (2) Animal preparation

 KKAy mice (CLEA Japan) were used as hyperglycemic (type I type I diabetes model) mice. In the feeding test, the ingredients weighed so as to have the ratios shown in Table 1 above were mixed, kneaded with distilled water, and then dried in a freeze dryer. (The mice fed with the food were referred to as “control group” in this example). In addition, the dry powder of hot water extract obtained in (1) above was added to the control food so that it would be 1%, and this was added to the Nemebiwa tea group (the mice fed this food were In the example, it was used as “biso tea group”.

-(3) Measurement of blood glucose level in type I diabetes model mice

 I Experiments were conducted using 20 KKAy mice (CLEA Japan) as type I diabetes model mice. The mice were divided into a control group (10 animals) and a bime tea group (10 animals), and the control group mice were allowed to freely ingest control food. In addition, mice in the bi ヮ cha group were given free access to Nemebiwa tea group food. Both groups were raised for 7 weeks each. Tap water was used in both groups for water supply to mice, and free water was provided during the breeding period. During the breeding period, blood glucose levels of mice were measured every other week. The enzyme electrode method was used for blood glucose level measurement.

 Figure 8 shows the changes in blood glucose levels during the breeding period. In Fig. 8, the black circles are the results for the control group mice, and the white circles are the results for the Biwacha group mice.

As shown in Fig. 8, blood glucose levels gradually increase in both groups of animals, and it can be seen that there is a difference in blood glucose levels from the first week of the experiment. That is, it can be seen that the increase in blood glucose level was suppressed in the bi ヮ cha group mice compared to the control group mice. In particular, blood glucose levels at 3, 5, and 6 weeks were compared to the control group of mice. It can be seen that the sugar level is significantly lower (in Fig. 8, * indicates P is 0.05). (4) Glucose tolerance test in type II diabetes model mice

 After the rearing in (3) above, each group of mice was fasted for 14 hours, and glucose was administered thereto. The dose of glucose was 4 g / 10 ml of glucose (distilled water) with a mouse body weight of xl / 200 (ml). This dose was injected directly into the stomach of each mouse with a stainless steel sonde and forcibly administered. After administration, blood glucose levels were measured over time.

 After the measurement, the mouse was anesthetized with Nembutal, blood was collected from the heart, and the value of HbAlC was measured.

 Figure 9 shows the results of the glucose tolerance test after breeding. In Fig. 9, the black circles are the results for the control group of mice, and the white circles are the results for the Biwacha group of mice. In Fig. 9, * indicates P is 0.05.

 As shown in Fig. 9, blood glucose levels rose rapidly in both groups after glucose administration, reaching a high value of approximately 400 mg / dl after 15 minutes. After 30 minutes, it can be seen that the blood glucose level of the control group mice continued to rise and then declined. On the other hand, the blood glucose ^ S of the mice in the bi ヮ tea group was kept lower than that in the control group after 30 minutes (there was a significant difference between the P and 0.05 levels). It can be seen that the blood glucose level decreases as well as the value of the control group. In addition, it can be seen that the blood glucose level of the mice in the bi 群 tea group at each measurement time always keeps a lower value than the blood glucose level of the control group.

 Furthermore, the measurement results of blood HbAlC values after the glucose tolerance test are shown in FIG. In FIG. 10, “control” is the result of the mice in the control group, and “bi ヮ cha” is the result of the mice in the bi ヮ tea group.

 As shown in Fig. 10, the blood HbAlC value of mice in the BY tea group was significantly higher than the blood HbAlC value of mice in the control group (in Fig. 10, ** is P + 0.01). It shows that it is low. '

 [Example 3] Inhibitory effect of crude blood fraction of Nemebiwa tea on increase in blood glucose level

(1) Preparation of crude fraction of Nemebiwa tea

Commercial Nemebiwa tea (50 g) was extracted with hot water (400 ml). This was repeated three times. The resulting hot water extract was freeze-dried (Tokyo Rikakikai Co. 'LTD, Freeze Dryer FR-1) The dried powder was obtained. The total dry weight of Nemebiwa tea used was 950.0 g.

 The obtained hot water extract was cooled and then sequentially applied to an open column chromatogram of MCI gel CHP-20P. The amount of gel is preferably about 1 kg as a dry weight. The glass column used had an inner diameter of 8 cm. In this embodiment, a glass column is used, but the material of the column is not limited to glass, acrylic material, or the like. The solvent is eluted with water (2L), 30% aqueous ethanol (2L), 70% aqueous ethanol (2L) and water-acetonone (1: 1 ratio) (2L) to obtain 4 fractions. It was. The yield of the four dry crude fractions was 90.0 g of the dry crude fraction eluted with water (2 L) (hereinafter referred to as “Fraction 1”) and the dry crude fraction eluted with 30% aqueous ethanol (2 L). 45.3 g of the dry crude fraction (hereinafter referred to as “Fraction 2”) and 18.3 g of the dry crude fraction (hereinafter referred to as “Fraction 3”) eluted with 70% aqueous ethanol (2 L). The dry crude fraction (hereinafter referred to as “Fraction 4”) eluted with water-acetone (1: 1 ratio) (2 L) was 2. lg.

A thin layer chromatograph (Thin Layer Chromatography, hereinafter referred to as “TLC”) of these fractions 1 to 4 is shown in FIG. In Fig. 11, numbers 1 to 4 indicate the results of fractions ί to 4 respectively. The developing solvent used for TLC was benzene-ethyl formate-formic acid, and the ratio was mixed at a solution dose of 3: 6: 1. The coloring reagent used was a solution of about 5% ferric chloride (FeCl ₃ ) dissolved in methanol, and the compound was detected by color spraying directly onto TLC.

 As shown in Fig. 11, it can be seen that fractions 1 and 2 contain a chlorogenic acid-like substance in the vicinity of an Rf value of about 0.1. It can also be seen that fractions 3 and 4 do not contain much organic and chlorogenic acid-like substances.

(2) Preparation of animals-KKAy mice (CLEA Japan) were used as hyperglycemic (type II diabetes model) mice. In the feeding test, the ingredients weighed so as to have the ratios shown in Table 1 above were mixed, kneaded with distilled water, and then dried in a freeze dryer. Mice fed with the food were used as “control group” in this example. In addition, Fraction 1, Fraction 2 or Fraction 3 and 4 obtained in (1) above. Was added to the control bait to 1% each, and used as Nemebiwa tea group bait (the mice fed with the bait respectively were collectively referred to as the “boiled tea group” in this example). .

 (3) Measurement of blood glucose level in type I diabetes model mice

 I Type I diabetes model KKAy mice (CLEA Japan) were used in 32 experiments. Give mice a control group (8 animals), feed for Nemebiwa tea group containing fraction 1 Bean tea group (eight animals), bean meal give food for Nemebiwa tea group containing fraction 2 Divided into 4 groups: 8 tea groups, Biso tea group (8 animals) fed with Nemebiwa tea group containing a mixture of fractions 3 and 4, and 1 day for mice in the control group At one time, they were fed 1,5g of control food, and the shortage was fed with mouse sales. In addition, mice in each bi ヮ cha group were fed once a day with 1.5g of control food and 15mg of each fraction of Nemebiwa tea group feed. Was given a state of satiation. Each group was raised for 7 weeks. For each group, tap water was used for water supply and free water was used during the breeding period.

 During the breeding period, blood glucose levels of mice were measured every other week. The enzyme electrode method was used for blood glucose level measurement.

 Figure 12 shows the changes in blood glucose levels during the breeding period. In Fig. 1 2, the black circles are the results for the mice in the control mouth group, and the white squares are the mice from the Biwacha group that have been fed the Nemebiwa tea group food containing fraction 1. Is the result of The white triangles are the results for the bime tea group mice fed with the food for screw mebiwa tea group containing fraction 2, and the white circles are the screws containing fractions 3 and 4. This is the result of the mouse of the bime tea group fed with food for mebiwa tea group.

 As shown in Fig. 12, during the breeding period, the blood glucose level of the mice in the control group and each of the bicha tea groups increased. However, it can be seen that the blood sugar level of the bi ヮ tea group fed with fractions 3 and 4 peaked at 3 weeks after the start of the experiment, and remained low compared to the blood sugar level of the control group.

 (4) I glucose tolerance test in type I diabetes model mice

After the rearing in (3) above, each of the 4 groups of mice was fasted for 14 hours and glucose was administered thereto. The dose of buducose is 4g / 10ml of buducose (steamed) with mouse body weight X 1/200 (ml). Retained water). This dose was injected directly into the stomach of each mouse with a stainless steel sonde and forcibly administered. After administration, blood glucose levels were measured over time.

 After the measurement, the mouse was anesthetized with Nembutal, blood was collected from the heart, and the value of HbAlC was measured.

 Fig. 13 shows the results of the glucose tolerance test conducted on the Biwacha group fed with fractions 3 and 4 after the rearing. In Figure 1 3, the black circle is the result of the control group of mice, and the white circle is the mouse of the Biwacha group fed with the Nemebiwa tea group containing fractions 3 and 4. Is the result of

 As shown in Fig. 13, the blood glucose level of the bi ヮ cha group fed with fractions 3 and 4 was increased rapidly immediately after administration by glucose administration, and about 500 mg after 15 minutes. It rose to / dl. It can be seen that after 30 minutes, the control group continues to rise and then declines. On the other hand, it can be seen that in the green tea group fed with minutes 3 and 4, the blood glucose level began to decrease after 30 minutes and then decreased rapidly. In particular, after 30 and 60 minutes, it can be seen that the value is significantly lower (in Fig. 13, '* indicates P is 0.05).

 Furthermore, the measurement results of blood HbAlC levels after the glucose tolerance test are shown in FIG. In Fig. 14, “Control” is the result of the control group of mice, and “Biwacha 1” is the result of the Biwacha group fed with the Nemebiwa tea group containing fraction 1. Mouse results. In addition, it is the result of the mice of the Biwacha group that was fed with the feed for Nemebiwa tea group containing Fraction 2, and the Bibicha (3 + 4) force fraction 3 4 is a result of mice of the bi ヮ cha group fed with Nemebiwa tea group food containing 4 and 4.

 As shown in Fig. 4, the blood HbAlC value of mice in the bicha tea group fed with fractions 3 and 4 was significantly higher than the blood HbAlC value of mice in the control group (in Fig. 14, * Is P 0.

(Does not indicate 05) ..

[Example 4] Effect of antioxidant action of crude fraction of Nemebiwa tea

 (1) Preparation of crude fraction of Nemebiwa tea

Commercial Nemebiwa tea (1247 g) was extracted with 80% aqueous acetone (5000 ml). This was repeated three times. From the obtained 80% aqueous acetone extract, the solvent was distilled off under reduced pressure to obtain a syrupy extract (448.2 g). Part of the resulting extract (278g) was collected And subjected to column chromatography of MCI gel CHP-20P. The amount of gel is preferably about 1 kg as dry weight. The glass column used had an inner diameter of 8 cm. In this embodiment, a glass column is used, but the material of the column is not limited to glass or acrylic material. Chromatography was performed by using a linear gradient from solution A to solution B using water as the solution A and methanol as the solution B as the mobile phase. More specifically, crude fraction 1 was obtained by combining fraction solutions eluted from liquid A and liquid B at a ratio of 1: 0 (300 ml) and 95: 5 (300 ml). The crude fraction 2 was obtained by combining the fraction solutions eluting A liquid and B liquid at 9: 1 ratio (300 ml), 8: 2 ratio (300 ml) and 7: 3 ratio (300 ml). did. The fraction solution obtained by eluting the A solution and the B solution at a ratio of 6: 4 (300 ml) was designated as crude fraction 3. The fraction solution obtained by eluting the A solution and the B solution at a ratio of 5: 5 (300 ml) was designated as crude fraction 4. The fraction solution obtained by eluting the A solution and B solution at a ratio of 4: 6 (300 ml) was designated as crude fraction 5. The fraction solution obtained by eluting the A solution and the B solution at a ratio of 3: 7 (300 ml) was designated as crude fraction 6. The fraction solution in which A solution and B solution were eluted at a ratio of 2: 8 (300 ml) was designated as crude fraction 7. The fraction solution in which A liquid and B liquid were eluted at a ratio of 1: 9 (300 m 1) was designated as crude fraction 8. The fraction solution obtained by eluting the A solution and the B solution at a ratio of 0: 1 (300 ml) was designated as crude fraction 9. Finally, acetone was used as the liquid C, and the chromatography was performed with the ratio of the liquid A and the liquid C being 50:50 (1 L). In addition, the ratio of this A liquid and C liquid was made into 50:50 (1L), and a mouth-to-mouth chromatography was performed, and the fraction solution which can be obtained was mixed with said crude fraction 9. As a result, nine crude fractions referred to as crude fractions 1 to 9 were obtained. The yield of the nine crude fractions is 12.3 g for crude fraction 1, 12.8 g for crude fraction 2, 0.4 g for crude fraction 3, 2.9 _g for crude fraction 4, and crude fraction 5 was 8.3 g, crude fraction 6 was 6.7 g, crude fraction 7 was 19.9 g, crude fraction 8 was 5.0 g, and crude fraction 9 was 4.9 g.

Of these nine crude fractions, the TLCs of crude fractions 2-8 are shown: Shown in 5. In FIG. 15, numbers ₂ to 8 indicate the results of crude fractions 2 to 8, respectively. The developing solvent used for TLC was three types of solvents: benzene-ethyl formate-formic acid, and the ratio was mixed at a solution volume of 3: 6: 1. The coloring reagent used was a solution in which about 5% of ferric chloride (FeCl ₃ ) was dissolved in methanol, and the compound was detected by color spraying directly onto TLC. As shown in Fig. 15, it can be seen that the substance that develops color from green to blue is a phenolic substance.

(2) Antioxidant action experiment

 The antioxidant activity experiment was performed using the method described in the following literature (Harwat, K. S. M. et al., Free Radical Res., 36: 177-187, 2002).

 The sample was prepared by adjusting each of the nine crude fractions obtained in (1) above to a concentration of 10 g / ml. As a control, dry powder of hot water extract of Biso tea was used at a concentration of 10 g / ml. As a prepared product, a standard product prepared by adjusting vitamin C (ascorbic acid) at a concentration of 5 g / m 1 and vitamin E adjusted at a concentration of S / zg / ml was used. Place these samples in a flat-bottom plate (96-well), add DPPH (1, 1-dipheny l-2-picrylhydrazyl, adjusted to the concentration of micromolar) 190 μΐ and mix for 10 seconds. , Protected from light and left for 30 minutes. After this reaction, the reaction solution of each well was measured with a microplate reader using absorbance at a wavelength of 490 nm, and the Trolox concentration was converted from the absorbance value using a Trolox absorbance standard curve. From the converted value, the radical scavenging rate was calculated from the Trolox radical scavenging standard curve. Figure 16 shows the experimental results of the antioxidant activity. In Figure 16 the sample numbers 1-9 correspond to the crude fractions 1-9, respectively. Sample No. 10 is a dry powder of hot water extract of bean tea. VC is vitamin C, and VE is vitamin E. As shown in Figure 16, it can be seen that crude fractions 6, 8 and 9 have a strong antioxidant effect. The degree is about the same as or higher than vitamin E, and about half that of vitamin C. In addition, it can be seen that the antioxidant effect of crude fractions 6, 8 and 9 is about 3 times stronger than the dry powder of hot water extract of bi ヮ cha.

 [Example 5] Effect of antioxidant action of crude fraction of Nemebiwa tea 2

(1) Antioxidant action experiment of fractions 1 to 4 prepared in Example 3-Antioxidant action experiment was carried out using the method described in the following literature (Harwat, KSM et al., Free Radical Res., 36: 177-187, 2002).

Samples were the four crude fractions obtained in Example 3; fractions 1 to 4 were each adjusted to a concentration of 10 β g / ml, and Nejimebiwa tea obtained in Example 1 as a control. the dry powder of hot water extract used was adjusted at a concentration of 10 / _g / _m l. These samples were applied to a flat-bottomed plate (96wel l) [1 and DPPH (1, l_diphenyl-2-picrylhydrazyl, adjusted at the concentration of the mouth mouth mora) to 190 μ1. In addition, after mixing for 10 seconds, it was left for 30 minutes in the dark. After this reaction, the reaction solution of each well was measured with a microplate reader using the absorbance at a wavelength of 490 nm, and the Trolox concentration was converted from the absorbance value using the Trolox absorbance standard curve. From the converted values, the radical scavenging rate was calculated from the Trolox radical scavenging standard curve.

 Figure 17 shows the experimental results of the antioxidant activity. In Fig. 17, the vertical axis represents DPPH radical scavenging rate as%, and the horizontal axis fraction 1 to fraction 4 samples are the fractions 1 to 4 prepared in Example 3, respectively. Correspond. Bi ヮ cha is a dried powder of Nemebiwacha hot water extract obtained in Example 1.

 As shown in Fig. 17, it can be seen that Fraction 2, Fraction 3 and Fraction 4 have a strong antioxidant effect. It can be seen that the degree is greater than the effect of Nemebiwa tea hot water extract obtained in Example 1. In addition, it can be seen that the antioxidant effect of Fraction 3 is about 1.6 times stronger than the dry powder of Nemebiwa Chatoyu extract obtained in Example 1.

• (2) Preparation of crude fraction of Nemebiwa tea

A dry powder of fraction 3 obtained in Example 3 (5.0 g) was weighed, dissolved in an appropriate amount of water, and subjected to open column chromatography. Open column black Matogurafi one condition, chroma Trex ODS stationary phase (Chromatorex 0DS, Fuji Shirishia Chemical Co., Fuj i Si lysia Chemical LTD. ) Using distilled water as the A solution mobile phase (H ₂ 0) Methanol (MeOH) was used as the B liquid, and various chromatographies were performed by increasing the content of the B liquid from the A liquid. Specifically, the solvent is 80%

H ₂ 0-20% MeOH (400 ml), 70% H ₂ 0-30% MeOH (400 ml), 60% H ₂ 0-40% MeOH (400 ml),

_{50% H 2 0-50% Me0H (} 400ml), 40% ¾0-60% MeOH (400ml), 30% H 2 0-70% Me0H (400 ml), 20% H 2 0-80% Me0H (400ml) Elution with 10% H ₂ 0-90% MeOH (400 ml), 100% MeOH (400 ml), fractionate each 400 ml elution fraction, and extract a total of 9 fractions from 3-1 to 3-9 Obtained. The yield and yield of each fraction were as follows. Fraction 3-1 (0.19g, 3.9%), fraction

3-2 (0.28g, 5.7%), Fraction 3-3 (0.57g, 11.6%), Fraction 3-4 (085g, 17.3%), Fraction 3- 5 (0.664g, 13.1%), Fraction 3-6 (0.79g, 16.1%), Fraction 3-7 (0.54g, 11.0%), Fraction 3-8 (0, 78g, 15.9%), Fraction 3-9 (0.17g, 3.5%).

 (3) Experiment of antioxidant action of fractions 3-1 to 3-9 further obtained from fraction 3 of Example 3 The experiment of antioxidant action was performed using the method described in the following literature ( Harwat, KSM et al., Free Radical Res., 36: 177-187, 2002).

 Samples were obtained in Example 3 as a control and the 9 crude fractions obtained in (2) above: fractions 3-1 to 3-9 adjusted to a concentration of 10 μg / ml, respectively. A dry powder prepared from fraction 3 at a concentration of 10 μg / ml was used.

 Add these samples to a flat-bottom plate (96wel l), and then add 190μ (of DPPH (1, 1-dipheny l-2-pi crylhydrazyl, adjusted to the concentration of micromolar) and mix for 10 seconds. , Protected from light and left for 30 minutes. After this reaction, the reaction solution of each well was measured with a microplate reader using the absorbance at a wavelength of 490 nm, and the Trolox concentration was converted from the absorbance value using the Trol ox absorbance standard curve. From the converted value, the radical scavenging rate was calculated from Trolox's standard curve for radical scavenging ability. Figure 18 shows the experimental results of the antioxidant activity. In Fig. 18, the vertical axis indicates DPPH radical scavenging rate as a percentage, and the horizontal axis 3-1 to 3-9 shows the fractions prepared for each of the prepared fractions 3-1 to 3-9. Correspond. Fraction 3 is Fraction 3 prepared in Example 3.

 As shown in Fig. 18, it can be seen that fractions 3-1, 3-2 and 3-4 have a strong antioxidant effect. It can be seen that the degree is larger than the effect of fraction 3 prepared in Example 3. In particular, it can be seen that the antioxidant effect of fraction 3-1 is about 1.37 times stronger than the dry powder of fraction 3.

 [Example 6] Effect of Nemebiwa tea extract on hypertension suppression

 (1) Animal preparation

SHR male rats (spontaneously hypertensive rats) (5 weeks old) ^two dogs I (manufactured by Nippon SLC Co., Ltd.) was used. In the feeding test, each material was weighed so as to have the ratio shown in Table 2 below, mixed, kneaded with distilled water, and then dried with a freeze dryer. It was used as a food (the rats fed with the food were called “control group” in this example). In addition, the dry powder of the hot water extract obtained in Example 1 was added to the control food so as to become 0.2%, and this was added to the food for Nemebiwa tea group (the rats fed with the food were used in this study). In the example, it was used as “biso tea group”. Table 2

(2) Measurement of rat blood pressure

SHR rats (rats with spontaneous hypertension) males (5 weeks old) (manufactured by SLC, Japan) were used to measure blood pressure using ^two I. These rats were divided into a control group (6 animals) and a bi ヮ cha group (6 animals), and the control group rats were allowed to freely ingest control food. Rats in the bi ヮ cha group were given free access to Nemebiwa tea group food. Both groups were reared for 50 days each.

 During the breeding period, feed and drinking water were freely consumed and freely drinking. The breeding room was 23 ° C and .12 hours lighting (7: 00-19: 00) '.

 After the breeding, in order to measure the blood pressure of each animal, an unheated force-type blood pressure measuring device (Muromachi Kikai Co., Ltd.) was used as a blood pressure measuring device.

The results of rat blood pressure measurements are shown in Figure 19. Figure 19 shows the changes in blood pressure in rats in both groups during the breeding period. In Fig. 9, the open circle is the label for the control group. The black circles are the results for the rats of the bi ヮ cha group. The blood pressure (maximum blood pressure) was measured from a certain time (around 10:00 in the morning). The measurement was repeated 10 times on average for each individual and expressed as the average value. Vertical bars indicate standard error. As an experimental difficulty in measuring blood pressure, blood pressure values fluctuate very easily, and can fluctuate very easily depending on the mental state and environmental conditions of the rat. Went under.

 As shown in Fig. 19, the average value of the maximum blood pressure in the control group and the bi-tea group was about 134 mmHg at the start. The blood pressure in the control group increased over 10 days with the passage of the breeding period, and remained fairly high after 20 days. In contrast, the Biwacha group did not increase until 21 days, and was significantly different from the control group on the 21st (P 〈0. 05). After that, the force S increased with the progress of the experiment days, and the blood pressure was lower by 10 mmHg or less than that of the control group, and the effect of Nejimewa tea extract on the suppression of hypertension was recognized. As a result, it can be seen that Nemebiwa tea extract suppresses an increase in blood pressure.

 [Example 7] Effect of the crude fraction of Nemebiwa tea on cancer cell proliferation inhibitory effect

'(1) Cancer cell proliferation inhibitory effect of fractions 1 to 4 prepared in Example 3'

 The 50% survival rate of human acute promyelocytic leukemia disease cells (HL-60 cells) was measured by the method described in the following literature (Mosmann, T., J. Immunol. Methods, 65: 55-63, 1983). ). HL-60 cells were cultured using RPMI1640 medium containing 10% fetal bovine serum. Concentrate 0, 25, 50, 75, 100, 125, 150, ug / ml of dry powder of Nemebiwacha hot water extract obtained in Example 1 and fractions 1-4 prepared in Example 3 As described above, HL-60 cells were treated with 0.1% DMS0 (dimethyl sulfoxide) solution and treated for 6 hours.

After the treatment, the cells were collected by centrifugation, the cell membrane was removed with a cell lysis buffer, RNA was degraded with RNase, and the protein was further removed with proteolytic enzyme. The obtained DNA fragments were separated by 2% agarose gel electrophoresis, stained and then detected by a UV transilluminator, and the DNA fragment rate was calculated. The 50% inhibitory concentration (IC ₅ ) was calculated for the HL-60 cell viability after 48 hours. The results are shown in Table 3. The extract in Table 3 is the result of dry powder of Nemebiwa tea hot water extract obtained in Example 1. Table 3

表 3に示すように、 ねじめびわ茶熱湯抽出液の乾燥粉末に対して、 分画 2〜4 は比較的強い HL- 60細胞増殖抑制効果を有した。中でも、分画 4は最も強い HL-60 細胞増殖抑制効果を有することが分かる。

As shown in Table 3, fractions 2-4 had a relatively strong HL-60 cell growth inhibitory effect on the dried powder of Nemebiwa-cha hot water extract. Among them, it can be seen that Fraction 4 has the strongest HL-60 cell growth inhibitory effect.

 (2) Cancer cell proliferation inhibitory effect of fractions 3-1 to 3-9 prepared in Example 5

 In the same manner as in (1) above, the inhibitory effect of fractions 3-1 to 3-9 prepared in Example 5 on HL-60 cell proliferation was examined.

 The 50% viability of HL-60 cells was measured by the method described in the following literature (Mo-smann, T., J. Immunol. Methods, 65: 55-63, 1983). HL-60 cells were cultured using RPMI1640 medium containing 10% fetal bovine serum. Dry powder of Nemebiwa tea hot water extract obtained in Example 1 and fractions 3-1 to 3-9 prepared in Example 5 were added at 0, 25, 50, 75, 100, 125, 150 μg. HL-60 cells were treated for 6 hours in 0.1% DMS0 (dimethyl sulfoxide) solution to contain at a concentration of / ml.

After the treatment, the cells were collected by centrifugation, the cell membrane was removed with a cell lysis buffer, RNA was degraded with RNase, and the protein was further removed with protease. The obtained DNA fragments were separated by 2% agarose gel electrophoresis, stained and then detected by a UV transilluminator, and the DNA fragment rate was calculated. The 50% inhibitory concentration (IC ₅ ) was calculated for the HL-60 cell viability after 48 hours. The results are shown in Table 4. The results for the dry powder of Nemebiwa-cha hot water extract are shown in Table 3. Table 4

As shown in Table 4, fractions 3-1, 3-2 and 3-4 had a relatively strong HL-60 cell growth inhibitory effect. Among them, it can be seen that Fraction 3-4 has the strongest HL-60 cell growth inhibitory effect.

 [Example 8] Effect of the crude fraction of Nemebiwa tea on apoptosis induction of cancer cells

(1) Cancer cell apoptosis-inducing effect of fractions 1 to 4 prepared in Example 3

 DNA fragmentation of human acute promyelocytic leukemia disease cells (HL-60 cells) was measured by the method described in the following literature (Hou, DX et al., Int. J. Oncol., 23: 705-712). , 2003). Fraction 2, fraction 3 and fraction 4 prepared in Example 3 were adjusted to three concentrations of 250, 500 and 750 g / ml, and HL-60 cells were treated for 6 hours to extract DNA from the cell nucleus. did. The obtained DNA was subjected to agarose gel electrophoresis, and the results are shown in FIG. 'In Fig. 20, M is a DNA marker, and C is a control at a concentration of O / ig / ml. As a result, DNA ladders were observed at concentrations of 500 ig / ml and 75'C ig / inl in fraction 3, indicating that apoptosis was induced.

 (2) Cancer cell apoptosis-inducing effect of fractions 3-1 to 3-9 prepared in Example 5

In the same manner as in (1) above, the cancer cell apoptosis-inducing effect of fractions 3-1 to 3-9 prepared in Example 5 was examined. The DNA fragmentation of HL-60 cells was measured by the method described in the following literature (Hou, DX et al., Int. J. Oncol., 23: 705-712, 2003). Each fraction 3-1 ～3- 9 prepared in Example 5 was adjusted to a concentration of 500 μ _§ / πι1, the HL-60 cells were treated for 6 hours, DNA was extracted from the cell nucleus. The obtained DNA was subjected to agarose gel electrophoresis, and the results are shown in FIG.

 In Fig. 21, Μ is a DNA marker and C is a control at a concentration of 0 / g / ml. As a result, DNA ladders were observed in fractions 3-1, 3-2, 3-4, and 3-6, indicating that apoptosis was induced. In particular, strong DNA ladders are observed in fractions 3-2 and 3-4, indicating that apoptosis is strongly induced. '

 [Example 9] Effect of reactive oxygen species producing action of crude fraction of Nemebiwa tea

(1) Reactive oxygen species production effect of fractions 1 to 4 prepared in Example 3

Human acute promyelocytic leukemia disease cells (HL-60 cells) were passaged at 2.0 x 10 ⁶ cel ls I ml. This was diluted 4-fold with 'RPMI1640 medium to give 2.0 × 10 ⁵ cells / 400 1. The required amount is 400 μΐ cell fluid per well, so 20 ml of cell fluid was prepared per plate. This was cultured for 24 hours at 37 ° C. in a 5% CO ₂ stream while gently shaking the plate to prevent cell bias. -

After culturing for 2 hours, fractions 1 to 4 prepared in Example 3 were added to this culture solution to a concentration of 500 1. For comparison, the extract of Nemebiwacha hot water prepared in Example 1 was similarly added. This was incubated for 15 minutes at 37 ° C, of 5% C0 ₂ incubator. After culturing, Mwel solution (5 mM DCFH-DA: 1 mg of DCFH-DA dissolved in 413 μ1 of ethanol; DCFA-DA, diglycol fluorescin 2 acetic acid) 1.6 μ 1 each dispensed, subsequently, they were incubated an additional 30 minutes at 37 ° C, 5% C0 ₂ under a stream of an incubator.

 After incubation, each well was measured with a microplate reader (excitation wavelength: 485 nm, emission wavelength: 530 nm), and compared to the control, the amount of reactive oxygen produced in HL-60 cells was determined from the absorbance. Calculated. The results are shown in Figure 22. The control is the result when the test was conducted with HL-60 cells only.

In Fig. 22, control represents a control. In FIG. 22, the extract is Nemebiwa tea hot water extract prepared in Example 1. As a result, Fraction 3 is HL-60 thin It can be seen that it has a strong ability to produce active oxygen in the vesicle.

 (2) Reactive oxygen species production effect of fractions 3-1 to 3-9 prepared in Example 5

 In the same manner as in (1) above, the effect of producing reactive oxygen species of fractions 3-1 to 3-9 prepared in Example 5 was examined.

HL-60 cells were passaged at 2.0 × 10 ⁶ cel ls / ml. This was diluted 4-fold with RPMI1640 medium to 2.0 × 10 ⁵ cells / 400 μ 1. The required amount was 400 1 cell solution per wel l, so 20 ml cell solution was prepared per plate. This was cultured for 24 hours at 37 ° C. in a 5% CO ₂ stream while gently shaking the plate to prevent cell bias.

After culturing for 24 hours, fractions 3-1 to 3_9 prepared in Example 5 were added to the culture solution so as to have a concentration of 500 μg / ml. This was incubated for 15 minutes at 37 ° C, of 5% C0 ₂ incubator. After culture, MTT solution (5 mM DCFH-DA: 1 mg of DCFH-DA dissolved in 413 μ 1 of ethanol; DCFA-DA, dichlorofluorescin 2 acetic acid) 1.6 / 1 each dispensed, subsequently, it was incubated 30 min further at 37 ° C, 5% C0 ₂ Inkyubeta one under a stream of air.

 After incubation, each well was measured with a microplate reader (excitation wavelength: 4.85 nm, ■ emission wavelength: 530 nm), and the amount of active oxygen produced in HL-60 cells was calculated from the absorbance compared to the control. did. The results are shown in Figure 23. The control is the result when the test was conducted with HL-60 cells only.

In Figure 23, chondrol indicates a control. As a result, it can be seen that fractions 3-3-2, 3-3 and 3-5 have a stronger ability to produce active oxygen than the control. In particular, it can be seen that Fraction 3-1 has about 2.6 times stronger active and reproductive oxygen producing capacity than HL-60 cells compared to the control. Industrial Applicability-According to the present invention, antihyperlipidemic action, antihypertensive action, cancer cell proliferation inhibitory action, cancer cell apoptosis inducing action, reactive oxygen species producing action, hyperglycemia lowering action and / or anti A food / beverage product or a pharmaceutical product having an oxidizing action and containing an extract or purified product of bime leaf or bime tea is provided. The food / beverage products (especially health supplements or foods for specified health use) or pharmaceutical products according to the present invention have an antihyperlipidemic action, an antihypertensive action, a cancer cell proliferation inhibiting action, Anti-hyperlipidemic agent, antihypertensive agent, cancer cell proliferation inhibitor, cancer cell apoptosis inducer, because it has a cell apoptosis inducing action, reactive oxygen species producing action, hyperglycemic lowering action and / or antioxidant action It can be used as a reactive oxygen species generator, a hyperglycemic agent, and an antioxidant.

 All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

The scope of the claims 1. Contains an extract or purified product of bime tea as an active ingredient, and has an antihyperlipidemic effect, an antihypertensive effect, an antihypertensive effect, an anticancer cell proliferation inducing effect, an active oxygen species producing effect, a high A food or drink or pharmaceutical having one or more actions selected from the group consisting of hypoglycemic action and antioxidant action.  2. The food or beverage or medicine according to claim 1, wherein the bean tea is Nemebiwa tea.  3. The purified product is selected from the group consisting of chlorogenic acid, quercetin 3-sambubioside, methyl chlorogenic acid, kaempferonole 3-rhamnoside, quenorecetin 3-rhamnoside, 2-hydroxyursolic acid and ursolic acid 1 The food or drink or pharmaceutical product according to claim 1, which does not contain the above compound.  4. The food or drink is selected from the group consisting of antihyperlipidemic action, antihypertensive action, cancer cell growth inhibitory action, cancer cell apoptosis inducing action, reactive oxygen species producing action, hyperglycemic lowering action and antioxidant action. It has an indication that it has one or more actions. The food / beverage product or pharmaceutical product according to claim 1, wherein  5. Inhibiting antihyperlipidemic action, antihypertensive action, cancer cell proliferation inhibitory action, cancer cell apoptosis, characterized in that it includes a step of subjecting bicha tea to hot water extraction or solvent extraction to obtain an extract. A method for producing a food or drink or pharmaceutical product having one or more actions selected from the group consisting of action, reactive oxygen species production action, hyperglycemic action and antioxidant action.  6. The production method according to claim 5, further comprising a step of subjecting the extract to a purification means to obtain a purified product. , 7. The production method according to claim 6, wherein the purification means is column chromatography. ..  8. The production method according to claim 5, wherein the bean tea is Nemebiwa tea. 9. The purified product is selected from the group consisting of chlorogenic acid, quercetin 3-sambubioside, methyl chlorogenic acid, kaempferonole 3-rhamnoside, quenorecetin 3-rhamnoside, 2-hydroxyursolic acid and ursolic acid 1 Above The production method according to claim 6, wherein the compound does not contain a compound.